Further indications of the multicomponent nature of the acrosome reaction-inducing substance of human follicular fluid

Human follicular fluid (hFF), which has been treated with either unspecific proteases or dextran-coated charcoal (DCC) to remove proteins and/or steroids, cannot successfully induce the acrosome reaction (AR). After the removal of steroids, AR-inducing activity can be restored to hFF by supplementation with exogenous progesterone, but only in the presence of intact protein. Gel filtration experiments with ³H-progesterone-labelled hFF showed elution of the radioactive signal in the high molecular weight range, corresponding to bound progesterone. AR-inducing activity was seen in exactly the same fraction. Based on these results, the acrosome reaction-inducing substance (ARIS) appears to be a complex of progesterone and a progesterone-binding protein, which was shown to be identical with the plasma protein corticosteroid-binding globulin (CBG) by immunological techniques. AR induction was only observed in the presence of both CBG and progesterone, suggesting a combined effect of the two components. © 1995 wiley-Liss, Inc.     

Inactivation of a Synechocystis sp strain PCC 6803 gene with homology to conserved chloroplast open reading frame 184 increases the photosystem II-to-photosystem I ratio.

A gene of the unicellular cyanobacterium Synechocystis sp strain PCC 6803 that is homologous to the conserved chloroplast open reading frame orf184 has been cloned and sequenced. The nucleotide sequence of the gene predicts a protein of 184 amino acids with a calculated molecular mass of 21.5 kD and two membrane-spanning regions. Amino acid sequence analysis showed 46 to 37% homology of the cyanobacterial orf184 with tobacco orf184, rice orf185, liverwort orf184, and Euglena gracilis orf206 sequences. Two orf184-specific mutants of Synechocystis sp PCC 6803 were constructed by insertion mutagenesis. Cells of mutants showed growth characteristics similar to those of the wild type. Their pigment composition was distinctly different from the wild type, as indicated by an increase in the phycocyanin-to-chlorophyll ratio. In addition, mutants also had a two- to threefold increase in photosynthetic electron transfer rates as well as in photosystem II-to-photosystem I ratio-a phenomenon hitherto not reported for mutants with altered photosynthetic characteristics. The observed alterations in the orf184-specific mutants provide strong evidence for a functional role of the orf184 gene product in photosynthetic processes.     

Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition.

Progression through the cell cycle is monitored at two major points: during the G1/S and the G2/M transitions. In most cells, the G2/M transition is regulated by the timing of p34cdc2 dephosphorylation which results in the activation of the kinase activity of the cdc2-cyclin B complex. The timing of p34cdc2 dephosphorylation is determined by the balance between the activity of the kinase that phosphorylates p34cdc2 (wee1 in human cells) and the opposing phosphatase (cdc25C). Both enzymes are regulated and it has been shown that cdc25C is phosphorylated and activated by the cdc2-cyclin B complex. This creates a positive feed-back loop providing a switch used to control the onset of mitosis. Here, we show that another member of the human cdc25 family, cdc25A, undergoes phosphorylation during S phase, resulting in an increase of its phosphatase activity. The phosphorylation of cdc25A is dependent on the activity of the cdc2-cyclin E kinase. Microinjection of anti-cdc25A antibodies into G1 cells blocks entry into S phase. These results indicate that the cdc25A phosphatase is required to enter S phase in human cells and suggest that this enzyme is part of an auto-amplification loop analogous to that described at the G2/M transition. We discuss the nature of the in vivo substrate of the cdc25A phosphatase in S phase and the possible implications for the regulation of S phase entry.     

Insect immunity. A transgenic analysis in Drosophila defines several functional domains in the diptericin promoter.

Diptericins are antibacterial polypeptides which are strongly induced in the fat body and blood cells of dipteran insects in response to septic injury. The promoter of the single-copy, intronless diptericin gene of Drosophila contains several nucleotide sequences homologous to mammalian cis-regulatory motifs involved in the control of acute phase response genes. Extending our previous studies on the expression of the diptericin gene, we now report a quantitative analysis of the contribution of various putative regulatory elements to the bacterial inducibility of this gene, based on the generation of 60 transgenic fly lines carrying different elements fused to a reporter gene. Our data definitively identify two Kappa B-related motifs in the proximal promoter as the sites conferring inducibility and tissue-specific expression to the diptericin gene. These motifs alone, however, mediate only minimal levels of expression. Additional proximal regulatory elements are necessary to attain some 20% of the full response and we suspect a role for sequences homologous to mammalian IL6 response elements and interferon-gamma responsive sites in this up-regulation. The transgenic experiments also reveal the existence of a distal regulatory element located upstream of -0.6 kb which increases the level of expression by a factor of five.     

The Na-K-2Cl cotransporter is in a permanently activated state in cytoplasts from Ehrlich ascites tumor cells

Brief incubation of Ehrlich ascites tumor cells with cytochalasin B causes the formation of blebs in the surface membrane. Gentle homogenization removes the blebs as intact cytoplasts which contain neither mitochondrian or nucleus, nor other cytoplasmic membranous organelles. The Na-K-2Cl cotransporter is present in the cytoplasts in a permanently activated state, whereas the Na-K-2Cl transport system in unperturbed intact cells is silent. Pretreatment of intact cells with cytochalasin B for l min stimulates the bumetanide-inhibitable K⁺ influx fivefold. The influx into purified cytoplasts when expressed per g protein is three- to fourfold higher than the influx into cytochalasin B-treated intact cells. Thus, the membrane vesicles are enriched with the cotransporter, and the cotransporter is present in an activated state. The K influx into cytoplasts is inhibited about 40% by Na-free, Cl-free or bumetanide-containing media and to a similar extent by Fab fragments prepared from antiserum against purified proteins of the cotransporter. The K _I for bumetanide was 0.19±0.06 m for the cytoplasts as compared to 0.67±0.11 m for the intact cells. SDS gel electrophoresis of membrane proteins from the cytoplast membranes compared to the membranes of intact cells shows a reduced number of bands and a majority of bands showing reduced staining, whereas a few bands are stained more intensely. Particularly notable is a band at 80 kD, which is similar to the molecular weight previously reported for the main membrane protein isolated from intact cells using a bumetanide-Sepharose affinity column. An immunoblot of the cytoplast preparation using antibodies against the purified bumetanide binding proteins showed strong immunodetection of the 80 kD protein.We are grateful to Marianne Schiødt, Birgit Blytmann Jørgensen, Thomas Krarup and Beverley Dyer for expert assistance. This work was supported by grants from the Danish Natural Science Research Council (11-6835 to E.K.H.) and the National Institutes of Health (DK 33640 to P.B.D.) and by a Carlsberg Foundation research fellowship (to F.J.).     

Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1

The goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 -induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.     

The effects of acclimation and rearing conditions on the response of tropical and temperate populations ofDrosophila melanogaster andD. simulans to a temperature gradient (Diptera: Drosophilidae)

The effects of rearing and acclimation on the response of adultDrosophila to temperature were investigated in a gradient.D. melanogaster flies preferred a higher mean temperature and were distributed over a wider range of temperatures thanD. simulans flies. Acclimating adults at different temperatures for a week did not influence the response of either species. Adults reared at 28°C as immatures had a lower mean preference than those reared at cooler temperatures, suggesting that flies compensated for the effects of rearing conditions. Adults from tropical and temperate populations ofD. melanogaster andD. simulans did not differ in the mean temperature they preferred in a gradient, suggesting little genetic divergence for this trait within species. The species differences and environmental responses may be related to changes in optimal physiological conditions for the flies.     

Low temperature cultivation — A step towards process optimisation

Adherent recombinant BHK cells were cultivated at temperatures between 30 and 37°C. Batch and repeated-batch-cultivations in a 2-litre bioreactor showed a significant influence on metabolism and cell growth. The low-temperature-cultivations showed a lower growth rate and a lower glucose consumption rate and, therefore, less lactate production. On the other hand, the maximum cell density and productivity seemed not to be affected by the temperature reduction.     

Membrane function and vascular reactivity

This communication examines the possibility that nitric oxide (NO) production by endothelial cells results from changes in cell membrane fluidity. Lysophosphatidylcholine (LPC) alters fluidity of the endothelial cell membranes causing vascular relaxation. Through membrane alterations LPC influences function of a number of membrane receptors and modulates enzyme activity. As a result of detergent action, lysophosphatidylcholine (LPC) causes activation of guanylate cyclase, stimulates syalytransferase and regulates protein kinase C activity. It has already been demonstrated that ionic detergents, such as Triton X-100 also cause vascular relaxation, possibly induced by NO production from endothelial cells. It is postulated that production of nitric oxide results from changes in membrane viscosity; this may represent a mechanism for its regulation in biological systems.     

Fusion protein mediated prodrug activation (FMPA) in vivo

A two component system, consisting of a fusion protein and an appropriate prodrug, suited to perform selective tumor therapy in vivo, is presented. The fusion protein, owing to its humanized carcinoembryonic antigen (CEA)-specific variable region, specifically binds to CEA-expressing tumors and has an enzymatic activity comparable to human β-glucuronidase. The prodrug is a nontoxic glucuronide-spacer-derivative of doxorubicin decomposing to doxorubicin by enzymatic deglucuronidation. In vivo studies in nude mice bearing human CEA-expressing tumor xenografts revealed that 7 d after injection of 20 mg/kg fusion protein, a high specificity ratio (>100:1) was obtained between tumor and plasma. Injection of 250 mg/kg of prodrug at d 7 resulted in tumor therapeutic effects superior to conventional chemotherapy without any detectable toxicity. These superior therapeutic effects that were observed using established human tumor xenografts can be explained by the approx 10-fold higher drug concentrations found in tumors of mice treated with fusion protein and prodrug than in those treated with the maximal tolerable dose of drug alone.